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January 15, 2009 (Vol. 29, No. 2)

Optimization of Binding Interaction Studies

New Approach Advocates Interspot Referencing Using Multiplexed SPR Arrays

  • Multiplexed SPR

    Click Image To Enlarge +
    Figure 2. The ProteOn interaction array

    An alternate approach utilizes Bio-Rad Laboratories’ ProteOn  XPR36 multiplexed SPR device, which integrates state-of-the-art microfluidics with a novel optical design. A specially designed Multi-Channel Module (MCM) is used to create a unique six channel by six channel interaction array on a sensor chip for the analysis of up to six ligands with panels of up to six analytes, producing 36 data points in a single experiment (Figure 2). Because multiple conditions can be tested in parallel, robust kinetic analysis of an analyte concentration series can be handled in one experiment. This One-shot Kinetics™ parallel approach can generate a complete kinetic profile of a biomolecular interaction, without the need for regeneration, in one experiment, using one injection of analyte and a single sensor chip.

  • Interspot Referencing

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    Figure 3. The ProteOn sensor chip surface

    The ProteOn XPR 36 protein interaction array system eliminates the need for a separate channel reference and allows all channels to be used to collect binding interaction data. The 6x6 interaction array pattern not only increases the number of interactions that can be monitored on a single chip, but it also creates unique interspot areas directly adjacent to the reaction spots (Figure 3). There are 36 interaction spots and 84 interspots, 42 vertical and 42 horizontal.

    In a typical ProteOn One-Shot Kinetics experiment the six vertical channels are activated and the ligand of interest is bound to them. The analyte of interest is then injected into the horizontal channels, and the binding response is measured for each interaction spot.

    This process gives rise to horizontal interspots that have not been exposed to the ligand or any of the reagents used to bind the ligand to the sensor chip. These interspots can be used to measure the nonspecific interaction of the analyte with the chip surface, bulk effects, and signal drift. The data are averaged from the two interspots adjacent to the interaction spot.

    The horizontal interspot signal is then subtracted from the reaction spot signal, providing the kind of reference-corrected binding data previously obtained with sequential flow cell designs. This eliminates the need to dedicate a flow cell or channel for use as a reference. In addition, the interspots provide accurate and reliable reference data due to their close proximity to the interaction spots.

    The vertical interspots can be used to provide a good measure of the uniformity of the immobilization of the ligand to the sensor chip, and to monitor the potential loss of a ligand that is not covalently bound to the sensor chip, such as an antibody captured by an immunoglobulin-binding protein (e.g., protein A).

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